We want to understand the molecular mechanisms of Ig class switch DNA recombination (CSR). CSR entails generation of double-strand DNA breaks (DSBs) and DSB resolution, as mediated by AID, Ung and other DNA-binding proteins/repair enzymes. In this grant current funding period (years 06-10), we have made important discoveries, including the: (i) identification of HoxC4 as trans-activator of AID gene promoter and demonstration of HoxC4 promoter activation by estrogen; (ii) characterization of 5'-AGCT-3' as the major con- served repeats in all IgH switch (S) regions and specific targets of 14-3-3 proteins; (iii) demonstration that 14-3- 3 function as DNA/protein adaptors that bind S regions/AID and PKA for CSR; (iv) identification of a post- cleavage role of AID in CSR; and (v) demonstration of an important role of the Rev1 DNA polymerase in CSR. Here, we want to identify the role of open chromatin in recruiting/stabilizing 14-3-3, AID, PKA, Rev1 and Ung to/on S regions, characterize the scaffolding functions of these factors in CSR and identify mechanisms of CSR inhibition. We hypothesize that 14-3-3, which have a high affinity for 5'-AGCT-3' repeats in all S regions, target only those that will undergo recombination due to the open chromatin state (reflected by germline transcription and combinatorial patterns of histone PTMs - our preliminary findings) of these regions, where they are stabilized by histone PTMs (Aim 1). Prompted by our preliminary findings that Rev1 recruits/stabilizes Ung to/on S regions, we contend that in addition to 14-3-3, which are bona fide scaffolding adaptors, AID, PKA, Rev1 and Ung possess scaffolding functions that reciprocally contribute to their recruitment/stabilization to/on S regions, and disruption of such scaffolding functions by naturally occurring or synthetic molecules aborts CSR (Aim 2). We will test our hypotheses by: (Aim 1.1) assessing the open chromatin state of the recombining S regions, including germline transcription and combinatorial patterns of histone PTM H3S10ph, H3K9/K14ac H3K4me3, H2BK120ub and H2BS14ph; (Aim 1.2) analyzing the selective binding of 14-3-3, AID, PKA, Rev1 and Ung to the recombining S regions, and its dependency on histone PTMs; (Aim 1.3) addressing the role of H3K9acS10ph, H3S10phK14ac and/or H2BK120ub PTMs in recruitment/stabilization of 14-3-3, Rev1 and AID to/on the recombining S regions; (Aim 2.1) defining 14-3-3, AID, PKA, Rev1 and/or Ung molecular interactions and their scaffolding functions that are central to CSR; (Aim 2.2) further understanding 14-3-3, AID, PKA, Rev1 and/or Ung scaffolding functions and interfering with such scaffolding functions to abort CSR by using naturally occurring Vpr/short Vpr peptides; (Aim 2.3) identifying, by high-throughput screening (HTS), synthetic small molecules that interfere with 14-3-3, AID, PKA, Rev1 and/or Ung interactions to abort CSR. Our experiments are timely and based on convincing preliminary data. They use innovative approaches, such as BiFC, qChIP, ChIP-seq, Seq-ChIP, RNA-seq, FRET, Vpr peptides, HTS and new KO/mutant mice, and they will significantly advance the development of therapeutics that block unwanted CSR, e.g., to IgG autoantibodies or atopic IgE.